Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 (Adgrf1).

Background: Recent research has shown that the adhesion G protein-coupled receptor F1 (Adgrf1; also known as GPR110; PGR19; KPG_012; hGPCR36) is an oncogene. The evidence is mainly based on high expression of Adgrf1 in numerous cancer types, and knockdown Adgrf1 can reduce the cell migration, invasion, and proliferation. Adgrf1 is, however, mostly expressed in the liver of healthy individuals. The function of Adgrf1 in liver has not been revealed. Interestingly, expression level of hepatic Adgrf1 is dramatically decreased in obese subjects. Here, the research examined whether Adgrf1 has a role in liver metabolism. Methods: We used recombinant adeno-associated virus-mediated gene delivery system, and antisense oligonucleotide was used to manipulate the hepatic Adgrf1 expression level in diet-induced obese mice to investigate the role of Adgrf1 in hepatic steatosis. The clinical relevance was examined using transcriptome profiling and archived biopsy specimens of liver tissues from non-alcoholic fatty liver disease (NAFLD) patients with different degree of fatty liver. Results: The expression of Adgrf1 in the liver was directly correlated to fat content in the livers of both obese mice and NAFLD patients. Stearoyl-coA desaturase 1 (Scd1), a crucial enzyme in hepatic de novo lipogenesis, was identified as a downstream target of Adgrf1 by RNA-sequencing analysis. Treatment with the liver-specific Scd1 inhibitor MK8245 and specific shRNAs against Scd1 in primary hepatocytes improved the hepatic steatosis of Adgrf1-overexpressing mice and lipid profile of hepatocytes, respectively. Conclusions: These results indicate Adgrf1 regulates hepatic lipid metabolism through controlling the expression of Scd1. Downregulation of Adgrf1 expression can potentially serve as a protective mechanism to stop the overaccumulation of fat in the liver in obese subjects. Overall, the above findings not only reveal a new mechanism regulating the progression of NAFLD, but also proposed a novel therapeutic approach to combat NAFLD by targeting Adgrf1. Funding: This work was supported by the National Natural Science Foundation of China (81870586), Area of Excellence (AoE/M-707/18), and General Research Fund (15101520) to CMW, and the National Natural Science Foundation of China (82270941, 81974117) to SJ.

Being overweight or obese increases the risk of developing numerous medical conditions including non-alcoholic fatty liver disease (NAFLD), where excess fat accumulates in the liver. NAFLD is a major global health issue affecting about 25% of the world's population and, if left untreated, can lead to liver inflammation as well as serious complications such as type 2 diabetes, heart disease, and liver cancer. Currently, there are no medications which specifically treat NFALD. Instead, only medications which help to manage the associated health complications are available. Therefore, a better understanding of NFALD is required to help to develop new strategies for diagnosing and treating the progression of this disease. A family of proteins known as GPCRs have crucial roles in regulating various bodily processes and are therefore commonly targeted for the treatment of disease. By identifying the GPCRs specifically involved in liver fat accumulation, new treatments for NFALD could be identified. Previous studies identified a GPCR known as Adgrf1 that is mainly found in liver cells, but its role remained unclear. To investigate the function of Adgrf1 in the liver, Wu et al. studied obese mice and human patients with NAFLD. The experiments showed that elevated levels of Adgrf1 in human and mouse livers led to increased fat accumulation. On the other hand, livers with lower levels of Adgrf1 exhibited reduced fat levels. A technique called RNA sequencing revealed that Adgrf1 induces expression of enzymes involved in fat synthesis, including a key regulator called Scd1. Treating mice with high levels of liver fat with molecules that inhibit Scd1 decreased the symptoms of Adgrf1-mediated fatty liver disease. These findings suggest therapies that decrease the levels of Adgrf1 may help to stop too much fat accumulating in the liver of human patients who are at risk of developing NAFLD. Further research is needed to confirm the effectiveness and safety of targeting Adgrf1 in humans and to develop suitable candidate drugs for the task.

Virus-Derived Chemokine Modulating Protein Pre-Treatment Blocks Chemokine-Glycosaminoglycan Interactions and Significantly Reduces Transplant Immune Damage.

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASONdst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss.

Targeted Delivery of Antisense Oligonucleotides Through Angiotensin Type 1 Receptor.

We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

Dopamine metabolite levels in the vitreous of diabetic and non-diabetic humans.

Animal studies suggest that the retinal dysfunction in diabetic subjects that precedes overt clinical vasculopathy may be due to a retinal dopamine deficit. We analyzed levels of dopamine (DA) and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the vitreous of diabetic and non-diabetic human subjects. Adult patients undergoing pars plana vitrectomy for non-hemorrhagic indications were prospectively recruited from the Emory Eye Center in Atlanta, GA. Vitreous samples were analyzed using high performance liquid chromatography (HPLC) to measure levels of DOPAC and DA in the vitreous specimens. Vitreous samples from 9 diabetic patients and 20 from non-diabetic patients were analyzed. No eyes had apparent diabetic retinopathy. Mean normalized DA concentration in vitreous of diabetic subjects was 0.76 ± 0.12 pg/µL vs. 0.73 ± 0.08 pg/µL in non-diabetic vitreous (p = 0.849). DOPAC concentration was 8.84 ± 0.74 pg/µL in vitreous of diabetic subjects vs. 9.22 ± 0.56 pg/µL in vitreous of non-diabetic subjects (p = 0.691). No difference was observed in the concentrations of DA and DOPAC in the vitreous of people without diabetes compared to those with diabetes without retinopathy.

EFFECT OF LUBRICANTS ON CORNEAL THICKNESS AFTER VITRECTOMY.

PURPOSE: This study examines the impact of corneal surface lubricants used during pars plana vitrectomy on corneal edema. METHODS: This prospective, observational, clinical study occurred at an academic institution. Participants were individuals aged 18 years and older who had already consented to undergo pars plana vitrectomy, without pre-existing corneal pathology. A corneal lubricant was chosen by the surgeon. Corneal thickness was measured preoperatively and postoperatively using pachymetry and anterior segment optical coherence tomography (AS-OCT). Main outcome measure was change in corneal thickness as measured by pachymetry. RESULTS: Forty-one patients completed the study protocol. The 23 subjects in the SHCS group had a significantly smaller increase in corneal thickness as measured by pachymetry compared with the 18 subjects in the HPMC group (29.9 µm vs. 58.1 µm, P value 0.02). When measured by anterior segment optical coherence tomography, the SHCS group had a smaller increase in corneal thickness compared with the HPMC group (0.04 mm vs. 0.06 mm, P value 0.09) but did not reach significance. CONCLUSION: SHCS is associated with reduced postoperative increase in corneal pachymetry as compared to HPMC.

Different blood pressure responses in hypertensive rats following chemerin mRNA inhibition in dietary high fat compared to dietary high-salt conditions.

Chemerin is a contractile adipokine, produced in liver and fat, and removal of the protein by antisense oligonucleotides (ASO) lowers blood pressure in the normal Sprague Dawley rat. In humans, chemerin is positively associated with blood pressure and obesity so we hypothesized that in a model of hypertension derived from high-fat (HF) feeding, the chemerin ASO would reduce blood pressure more than a high-salt (HS) model. Male Dahl S rats were given a HF (60% kcal fat; age 3-24 wk) or HS diet (4% salt; age 20-24 wk to match age and blood pressure of HF animals). Scrambled control, whole body, or liver-specific ASOs that knock down chemerin were delivered subcutaneously once per week for 4 wk with tissue and blood collected 2 days after the last injection. Conscious blood pressure was measured 24 h/day by radiotelemetry. By the end of whole body ASO administration, blood pressure of HF animals had fallen 29 ± 2 mmHg below baseline, while blood pressure of HS-diet animals fell by only 12 ± 4 mmHg below baseline. Administration of a liver-specific ASO to HF Dahl S resulted in a 6 ± 2 mmHg fall in blood pressure below baseline. Successful knockdown of chemerin in both the whole body and liver-specific administration was confirmed by Western and PCR. These results suggest that chemerin, not derived from liver but potentially from adipose tissue, is an important driver of hypertension associated with high fat. This knowledge could lead to the development of antihypertensive treatments specifically targeted to obesity-associated hypertension.

Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

Endothelial cell CD36 optimizes tissue fatty acid uptake.

Movement of circulating fatty acids (FAs) to parenchymal cells requires their transfer across the endothelial cell (EC) barrier. The multiligand receptor cluster of differentiation 36 (CD36) facilitates tissue FA uptake and is expressed in ECs and parenchymal cells such as myocytes and adipocytes. Whether tissue uptake of FAs is dependent on EC or parenchymal cell CD36, or both, is unknown. Using a cell-specific deletion approach, we show that EC, but not parenchymal cell, CD36 deletion increased fasting plasma FAs and postprandial triglycerides. EC-Cd36-KO mice had reduced uptake of radiolabeled long-chain FAs into heart, skeletal muscle, and brown adipose tissue; these uptake studies were replicated using [11C]palmitate PET scans. High-fat diet-fed EC-CD36-deficient mice had improved glucose tolerance and insulin sensitivity. Both EC and cardiomyocyte (CM) deletion of CD36 reduced heart lipid droplet accumulation after fasting, but CM deletion did not affect heart glucose or FA uptake. Expression in the heart of several genes modulating glucose metabolism and insulin action increased with EC-CD36 deletion but decreased with CM deletion. In conclusion, EC CD36 acts as a gatekeeper for parenchymal cell FA uptake, with important downstream effects on glucose utilization and insulin action.

Whole-Body but Not Hepatic Knockdown of Chemerin by Antisense Oligonucleotide Decreases Blood Pressure in Rats.

Chemerin is an inflammatory adipokine positively associated with hypertension and obesity. The majority of chemerin derives from the liver and adipose tissue, however, their individual contributions to blood pressure are unknown. We began studying chemerin in the normal rat using antisense oligonucleotides (ASO) with whole-body activity (Gen 2.5 chemerin ASO) or liver-restricted activity (GalNAc chemerin ASO). We hypothesized that in normotensive male Sprague-Dawley rats, circulating chemerin is predominately liver-derived and regulates blood pressure. A dosing study of the Gen 2.5 chemerin ASO (with a scrambled control ASO) supported 25 mg/kg as the appropriate dose. GalNAc chemerin ASO was also assessed and used at 10 mg/kg. Radiotelemetry monitored mean arterial pressure (MAP) for a 1-week baseline and weekly subcutaneous ASO injections for 4 weeks. Two days after the final injection, animals were euthanized for tissue reverse transcription-polymerase chain reaction and chemerin Western analysis. Gen 2.5 chemerin ASO treatments reduced chemerin mRNA and protein in liver, retroperitoneal fat (RP), and mesenteric perivascular adipose tissue (mPVAT), as well as reducing protein in plasma. GalNAc chemerin ASO treatments reduced chemerin mRNA and protein in liver and chemerin protein in plasma but had no effect on expression in RP fat or mPVAT. Gen 2.5 chemerin ASO treatment reduced MAP compared with control ASO but was unchanged in animals receiving the GalNAc chemerin ASO. Although circulating chemerin is liver-derived, it does not play a major role in blood pressure regulation. Local effects of chemerin from fat may explain this discrepancy and support chemerin's association with hypertension and obesity.

Blood Pressure Lowering and Safety Improvements With Liver Angiotensinogen Inhibition in Models of Hypertension and Kidney Injury.

Uncontrolled hypertension is an important contributor to cardiovascular disease. Despite the armamentarium of antihypertensive treatments, there remains a need for novel agents effective in individuals who cannot reach acceptable blood pressure levels. Inhibitors targeting the renin-angiotensin-aldosterone system (RAAS) are widely used but may not optimally inhibit RAAS and demonstrate an acceptable safety profile. Experiments were conducted to characterize a series of AGT (angiotensinogen) antisense oligonucleotides (ASOs) and compare their efficacy and tolerability to traditional RAAS blockade. AGT ASOs which target multiple systemic sites of AGT versus an N-acetylgalactosamine-conjugated AGT ASO that targets the liver were compared with captopril and losartan. Spontaneously hypertensive rats fed an 8% NaCl diet, a model of malignant hypertension resistant to standard RAAS inhibitors, demonstrated robust and durable blood pressure reductions with AGT ASO treatments, which was not observed with standard RAAS blockade. Studies in rat models of acute kidney injury produced by salt deprivation revealed kidney injury with ASO treatment that reduced kidney-expressed AGT, but not in animals treated with the N-acetylgalactosamine AGT ASO despite comparable plasma AGT reductions. Administration of either captopril or losartan also produced acute kidney injury during salt deprivation. Thus, intrarenal RAAS derived from kidney AGT, and inhibited by the standard of care, contributes to the maintenance of renal function during severe RAAS challenge. Such improvements in efficacy and tolerability by a liver-selective AGT inhibitor could be desirable in individuals not at their blood pressure goal with existing RAAS blockade.

Suppressing angiotensinogen synthesis attenuates kidney cyst formation in a Pkd1 mouse model.

Activation of the intrarenal renin angiotensin system (RAS) is believed to play an important role in the development of hypertension and cystogenesis in autosomal dominant polycystic kidney disease (ADPKD). Results of clinical studies testing RAS inhibitors in slowing the progression of cystic disease in ADPKD are inconclusive, and we hypothesized that current RAS inhibitors do not adequately suppress intrarenal RAS. For this study, we compared a novel Gen 2 antisense oligonucleotide (ASO) that inhibits angiotensinogen (Agt) synthesis to lisinopril in adult conditional Pkd1 systemic-knockout mice, a model of ADPKD. Six weeks after Pkd1 global gene knockout, the mice were treated with Agt-ASO (66 mg/kg/wk), lisinopril (100 mg/kg/d), PBS (control), or scrambled ASO (66 mg/kg/wk) for 10 wk, followed by tissue collection. Agt ASO resulted in significant reduction in plasma, liver, and kidney Agt, and increased kidney renin compared with control treatments. Kidneys from Agt-ASO-treated mice were not as enlarged and showed reduced cystic volume compared with lisinopril or control treatments. Blood pressure was better controlled with lisinopril than with Agt-ASO. Agt-ASO suppressed cell proliferation in both cystic and noncystic cells compared with lisinopril and control treatments. However, Agt-ASO did not reduce cell proliferation in liver, which indicates that Agt-ASO targets cell signaling pathways that specifically suppresses cystogenesis in the kidney. These data suggest that Agt-ASO effectively attenuates intrarenal RAS and therefore can be a novel and effective agent for treating ADPKD.

CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation.

Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.5 CD40 ASO reduced CD40 mRNA and protein levels 75-90% in the kidney. CD40 ASO treatment mitigated functional, transcriptional, and pathological endpoints of doxorubicin-induced nephropathy. Experiments using an activating CD40 antibody revealed CD40 is primed in kidneys following doxorubicin injury or unilateral ureter obstruction and CD40 ASO treatment blunted CD40-dependent renal inflammation. Suborgan fractionation and imaging studies demonstrated CD40 in glomeruli before and after doxorubicin administration that becomes highly enriched within interstitial and glomerular foci following CD40 activation. Such foci were also sites of ASO distribution and activity and may be predominately comprised from myeloid cells as bone marrow CD40 deficiency sharply attenuated CD40 antibody responses. These studies suggest an important role of interstitial renal and/or glomerular CD40 to augment kidney injury and inflammation and demonstrate that ASO treatment could be an effective therapy in such disorders.

Reduction of early reperfusion injury with the mitochondria-targeting peptide bendavia.

We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia's cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia's protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.

Sivelestat attenuates myocardial reperfusion injury during brief low flow postischemic infusion.

The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.

Hyperoxemic reperfusion after prolonged cardiac arrest in a rat cardiopulmonary bypass resuscitation model.

BACKGROUND: The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function. METHODS: Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups. RESULTS: At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group. CONCLUSIONS: One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.

Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion.

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.